pH dependence of copper geometry, reduction potential, and nitrite affinity in nitrite reductase
Artikel i vetenskaplig tidskrift, 2007

Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.

ACTIVE-SITE

ELECTRON-TRANSFER

INTRAMOLECULAR

ALCALIGENES-FAECALIS S-6

ATOMIC-RESOLUTION STRUCTURES

SUBSTRATE-BINDING

TYPE-2 COPPER

MOLECULAR REPLACEMENT

X-RAY-STRUCTURE

ACHROMOBACTER-CYCLOCLASTES

ANGSTROM RESOLUTION

Författare

Frida Jacobson

Chalmers, Kemi- och bioteknik, Molekylär bioteknik

A. Pistorius

Daniel Farkas

Göteborgs universitet

W. De Grip

Örjan Hansson

Göteborgs universitet

Lennart Sjölin

Göteborgs universitet

Richard Neutze

Göteborgs universitet

Journal of Biological Chemistry

0021-9258 (ISSN) 1083-351X (eISSN)

Vol. 282 9 6347-6355

Ämneskategorier

Kemiteknik

DOI

10.1074/jbc.M605746200

PubMed

17148448